The present invention relates to an apparatus and method for dispensing cells or particles confined in a free flying droplet, and in particular, to an apparatus and method appropriate for dispensing and/or printing a precisely defined number of cells or particles confined in a free flying droplet.
According to the state of the art, single cells can be detected, manipulated, and in particular, be sorted by means of flow cytometry (FCM). Flow cytometry is described by H. M. Shapiro, “Microbial analysis at the single-cell level: tasks and techniques, “Journal of Microbiological Methods, vol. 42, pp. 3-16, 2000. Flow cytometry is an established technology provided by various commercial companies and applied in many applications. Generally, a flow cytometer has five main components, a flow cell enabling a liquid stream, a measuring system, a detector and an analog-to-digital conversion (ADC) system, an amplification system and a computer for analysis of the signals. The flow cell enabling the liquid stream may use a sheath fluid and carries and aligns the cells so that they pass single file, i.e. one by one, through a light beam for sensing. The measuring system commonly uses measurement of impedance or conductivity or makes use of optical systems. Commonly used optical systems may comprise lamps (mercury, xenon), high-power water-cooled lasers (such as argon, krypton or dye lasers), low-power air-cooled lasers (such as argon lasers at a wavelength of 488 nm, red-HeNe lasers at a wavelength of 633 nm, green-HeNe lasers or HeCd lasers (UV)), diode lasers (blue, green, red, violet) for providing light signals. The detector and analog-to-digital conversion system generates Forward Scatter (FSC), Side Scatter (SSC) as well as fluorescence signals from light and converts them into electrical signals that can be processed by a computer. The amplification system may be linear or logarithmic.
FCM enables separation and sorting of single cells according to specific optical properties at high throughput. However, FCM is not able to deal with a very small sample volume (such as from 1 to 10 μl), because stationary flow conditions have to be established inside the cytometer. For the same reason, FCM cannot deliver a defined number of living cells in a small liquid aliquot with a volume of 100 nl or below. The sorting mechanism of FCM relies on a stationary flow inside the flow cell that cannot be switched on and off in a sufficiently short time.
There are also efforts in the art to miniaturize FCM into a smaller and more compact size. Lab on a chip (LOAC) flow cytometers (LOAC-FCM) were introduced which aim to provide a relatively low cost, small and compact FCM. Reference is made to K. Cheung, S. Gawad, and P. Renaud, “Impedance spectroscopy flow cytometry: On-chip label-free cell differentiation,” Cytometry Part A, vol. 65A, pp. 124-132, 2005 and U.S. Pat. No. 7,294,249 B2. U.S. Pat. No. 7,294,249 B2 discloses a microfluidic component and method in a fluid using a substrate having a channel for leading through individual particles for sorting particles in a fluid flow, in particular in a liquid flow. The component comprises a preparation area to specifically influence and separate the particles by means of dielectrophoresis, a measuring channel area having at least two sensing areas arranged in series with respect to the fluid flow direction, and a sorting area having electrode devices for sorting particles identified in the measuring channel area. Thus, U.S. Pat. No. 7,294,249 B2 discloses a miniaturized device for analyzing, counting an sorting cells or particles which do not need a labeling of cells. However, FCM technologies—whether standard or miniaturized—do not facilitate to locate selected cells or particles for advanced applications like single cell arrays or cell printing and are to be considered as continuous methods.
Recently, inkjet printing technology has been exploited to deliver living cells instead of inks for locating cells precisely into desired patterns, see T. Xu, J. Jin, C. Gregory, J. J. Hickman, and T. Boland, “Inkjet printing of viable mammalian cells,” Biomaterials, vol. 26, pp. 93-99, 2005, and S. Moon, S. K. Hasan, Y. S. Song, F. Xu, H. O. Keles, F. Manzur, S. Mikkilineni, J. W. Hong, J. Nagatomi, E. Haeggstrom, A. Khademhosseini, and U. Demirci, “Layer by Layer Three-dimensional Tissue Epitaxy by Cell-Laden Hydrogel Droplets,” Tissue Engineering Part C: Methods, vol. 16, pp. 157-166, 2010. In addition, reference is made to US 2009/0208577 A1. Inkjet printing technology enables much smaller volumes of aliquots, and at the same time, spatial resolved printing of cells confined in the droplets. Several applications have been demonstrated using this technology, especially in constructing artificial tissues or organs, arraying cells for high throughput cell screening in drug discovery, basic cell study and analysis. Inkjet printing technology confines cells in a liquid volume which is jetted in the form of a droplet in-flight, such that it offers a non-invasive or minimally invasive cell manipulation technique. Although the concept of printing cells suspended in free flying droplets has been presented before, the number of cells per droplet is generally random, see U. Demirci and G. Montesano, “Single cell epitaxy by acoustic picolitre droplets,” Lab on a Chip, vol. 7, pp. 1139-1145, 2007.
US 2008/0286751 A1 discloses a dispensing device for microfluidic droplets especially for cytometry. A main micro-channel extends between two first and second tanks and a homogenous or heterogeneous cellular suspension passes through the main micro-channel. A second micro-channel crosses the main micro-channel and comprises an ejection orifice. Upon generating a pressure wave in the second channel, a droplet may be ejected via the ejection orifice. Impedance measurements and/or optical analysis are used to measure properties of cells circulating in the main micro-channel. Cells or particles are identified according to pertinent characteristics, detected electrically and/or optically, in particular by criteria of size, cytoplasmic conductivity and/or membrane capacitance. Depending on the measurement results, the device can be programmed for parametering an ejection device case by case. When a particle which verifies specific criteria is detected, a pressure pulse is applied to the second channel and a droplet is ejected via the ejection orifice.
According to US 2008/0286751 A1, the cells or particles are supplied in a main channel, while the ejection orifice is arranged in a second channel. Thus, the cells or particles are supplied in a stream perpendicular to the dispenser opening, which needs additional microfluidic flow focusing elements and external flow control equipment like high precision pumps. This enhances the complexity of the whole apparatus and needs considerable volumes of cell suspension to prime the complete apparatus. The cross-flow in close vicinity of the orifice furthermore leads to the drawback that the orifice design is compromised with respect to the droplet generation process. In particular, the liquid volume enclosed between the orifice and the pressure source applied for actuation is larger than for most other dispensing devices according to the state of the art, and the cross-flow channels do provide additional escape paths for liquid to be expelled out of the orifice. The design requirements for the cross-flow design are therefore contradictory to an optimum orifice design for precise and efficient generation of small droplets.
Beside the standard technology cited above, M. Nakamura, A. Kobayashi, F. Takagi, A. Watanabe, Y. Hiruma, K. Ohuchi, Y. Iwasaki, M. Horie, I. Morita, and S. Takatani, “Biocompatible Inkjet Printing Technique for Designed Seeding of Individual Living Cells,” Tissue Engineering, vol. 11, pp. 1658-1666, 2005, and R. Tornay, V. Chapuis, V. Haguet, F. Chatelain, and P. Renaud, “Electrical Detection and Ejection of Beads in a One-Cell-Per-Drop Microdispenser,” presented at Solid-State Sensors, Actuators and Microsystems Conference, 2007, TRANSDUCERS 2007, International, 2007, also disclose producing micro droplets from a supplied cell suspension.
Inkjet printing of viable cells is disclosed in U.S. Pat. No. 7,051,654 B2. U.S. Pat. No. 4,318,480 relates to a method and apparatus for positioning a point of droplet formation in a jetting fluid of an electrostatic sorting device. Finally, DE 197 06 513 relates to a micro dosing device using a pressure chamber and a flexible membrane adjacent the pressure chamber.